Anti-glucagon antibodies

ABSTRACT

The present invention provides antibodies that bind to glucagon and methods of using the same. According to certain embodiments, the antibodies of the invention bind human GCG with high affinity. The antibodies of the invention may be fully human antibodies. The antibodies of the invention are useful for the treatment of various diseases or disorders characterized by elevated blood glucose levels, as well as other GCG-related disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C §119(e) of U.S.provisional application No. 62/050,889, filed Sep. 16, 2014, which isherein specifically incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies, and antigen-bindingfragments thereof, which specifically bind glucagon, compositionscomprising these antibodies and methods of use thereof, for example, forthe treatment of disorders responsive to the modulation of glucagonlevels, such as diabetic and other glucagon related metabolic disorders,and the like.

BACKGROUND

Glucagon is a 29 residue polypeptide hormone, which in cooperation withinsulin, mediates homeostatic regulation of the amount of glucose in theblood. Glucagon primarily acts by stimulating certain cells, forexample, liver cells, to release glucose when blood glucose levels fall.The action of glucagon is opposite to that of insulin, which stimulatescells to take up and store glucose whenever blood glucose levels rise.Glucagon is produced in the alpha cells of the pancreas, whereas insulinis secreted from the neighboring beta cells.

It is an imbalance of glucagon and insulin that may play an importantrole in several diseases, such as diabetes mellitus and diabeticketoacidosis. In particular, studies have shown that higher basalglucagon levels and lack of suppression of postprandial glucagonsecretion contribute to diabetic conditions in humans (Muller et al., NEng J Med 283: 109-115 (1970)).

It is believed that glucagon's effects on elevating blood glucose levelsare mediated in part by the activation of certain cellular pathwaysfollowing the binding of glucagon (GCG) to its receptor (designatedGCGR). GCGR is a member of the secretin subfamily (family B) ofG-protein-coupled receptors and is predominantly expressed in the liver.The binding of glucagon to its receptor triggers a G-protein signaltransduction cascade, activating intracellular cyclic AMP and leading toan increase in glucose output through de novo synthesis(gluconeogenesis) and glycogen breakdown (glycogenolysis) (Wakelam etal., Nature, (1986) 323:68-71; Unson et al., Peptides, (1989),10:1171-1177; and Pittner and Fain, Biochem. J. (1991), 277:371-378).

The action of glucagon can be suppressed by providing an antagonist,such as a glucagon antibody, such as those described herein. Suchantibodies may prove useful in lowering blood glucose levels in diabetesor in other conditions, such as stress hyperglycemia. Furthermore, bylowering glucose levels, it may be possible to prevent or amelioratecertain of the long-term complications associated with elevated glucoselevels in diabetic patients.

Anti-glucagon antibodies are mentioned, e.g., in U.S. Pat. Nos.4,206,199; 4,221,777; 4,423,034; 4,272,433; 4,407,965; 5,712,105;WO2007/124463 and WO2013/081993. Nonetheless, there is a need in the artfor novel glucagon antagonists, such as the anti-glucagon antibodiesdescribed herein, for lowering blood glucose levels in patientssuffering from diabetes and other disorders associated with elevatedglucagon levels.

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibodies and antigen-binding fragmentsthereof that bind specifically to glucagon (GCG) and neutralize itsactivity.

In a first aspect, the invention provides isolated monoclonal antibodies(mAbs) and antigen-binding fragments thereof that bind to glucagon andinhibit or block its activity, for example, block the binding ofglucagon to its receptor, thereby blocking the elevation of bloodglucose levels. The antibodies or antigen binding fragments thereof maybe useful for lowering blood glucose levels in a subject that suffersfrom a disease or condition characterized in part by increased bloodglucose levels, such as diabetes mellitus, or stress hyperglycemia. Theantibodies may also be used to treat a wide range of conditions anddisorders in which blocking the interaction of glucagon with theglucagon receptor is desired, thereby having a beneficial effect. Theantibodies may ultimately be used to prevent the long-term complicationsassociated with elevated blood glucose levels in diabetic patients, orto ameliorate at least one symptom associated with elevated bloodglucose levels in diabetic patients.

In one embodiment, the invention provides an isolated monoclonalantibody or antigen-binding fragment thereof that binds specifically toand neutralizes GCG activity, wherein the antibody or fragment thereofexhibits one or more of the following characteristics:

(a) is a fully human monoclonal antibody;

(b) binds human GCG at 25° C. with a K_(D) of less than about 1 nM andless than about 5 nM at 37° C. as measured by surface plasmon resonance;

(c) lowers blood glucose levels by at least about 10% when administeredto a mammal as a single dose, or as multiple doses of less than about 40mg/kg;

(d) inhibits GCG-mediated activation of cells expressing human glucagonreceptor (GCGR) with an IC₅₀ of less than about 1 nM;

(e) comprises a heavy chain variable region (HCVR) having an amino acidsequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50,66, 82, 98, 114, 130 and 146; or

(f) comprises a light chain variable region (LCVR) having an amino acidsequence selected from the group consisting of SEQ ID NO: 10, 26, 42,58, 74, 90, 106, 122, 138 and 154.

In one embodiment, the antibody or antigen-binding fragment thereofbinds human GCG at 25° C. with a K_(D) ranging from about 50 pM to about750 pM and from about 300 pM to about 5 nM at 37° C. as measured bysurface plasmon resonance.

In one embodiment, the antibody or antigen-binding fragment thereoflowers blood glucose levels by about 10% to about 35% when administeredto a mammal as a single dose, or as multiple doses ranging from about 3mg/kg to about 30 mg/kg.

In one embodiment, the antibody or antigen-binding fragment thereofinhibits GCG-mediated activation of cells expressing human glucagonreceptor (GCGR) with an IC₅₀ ranging from about 7 pM to about 950 pM.

In one embodiment, the antibody or antigen-binding fragment thereofreduces blood glucose levels in a mammal when administered at a dose ofabout 30 mg/kg every 1, 2, 3, 4 or 5 days.

In one embodiment, the antibody or antigen-binding fragment thereofreduces blood glucose levels in a mammal when administered at a dose ofabout 30 mg/kg every 5 days.

In one embodiment the isolated antibody or antigen-binding fragmentthereof reduces blood glucose levels in a mammal when administeredsubcutaneously, intravenously, or intramuscularly.

The antibodies of the invention can be full-length (for example, an IgG1or IgG4 antibody) or may comprise only an antigen-binding portion (forexample, a Fab, F(ab′)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions (Reddy etal., 2000, J. Immunol. 164:1925-1933).

Exemplary anti-glucagon (anti-GCG) antibodies of the present inventionare listed in Tables 1 and 2 herein. Table 1 sets forth the amino acidsequence identifiers of the heavy chain variable regions (HCVRs), lightchain variable regions (LCVRs), heavy chain complementarity determiningregions (HCDR1, HCDR2 and HCDR3), and light chain complementaritydetermining regions (LCDR1, LCDR2 and LCDR3) of the exemplary anti-GCGantibodies. Table 2 sets forth the nucleic acid sequence identifiers ofthe HCVRs, LCVRs, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of theexemplary anti-GCG antibodies.

The present invention provides antibodies or antigen-binding fragmentsthereof that specifically bind GCG, comprising an HCVR comprising anamino acid sequence selected from any of the HCVR amino acid sequenceslisted in Table 1, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind GCG, comprising an LCVRcomprising an amino acid sequence selected from any of the LCVR aminoacid sequences listed in Table 1, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity thereto.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind GCG, comprising an HCVR and anLCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVRamino acid sequences listed in Table 1 paired with any of the LCVR aminoacid sequences listed in Table 1. According to certain embodiments, thepresent invention provides antibodies, or antigen-binding fragmentsthereof, comprising an HCVR/LCVR amino acid sequence pair containedwithin any of the exemplary anti-GCG antibodies listed in Table 1.

In one embodiment, the present invention provides an isolated antibodyor antigen-binding fragment thereof that specifically binds to GCG andneutralizes at least one activity associated with GCG, wherein theantibody or antigen-binding fragment thereof comprises: (a) three heavychain complementarity determining regions (HCDR1, HCDR2 and HCDR3)contained within a heavy chain variable region (HCVR) amino acidsequence selected from the group consisting of SEQ ID NOs: 2, 18, 34,50, 66, 82, 98, 114, 130 and 146; and (b) three light chain CDRs (LCDR1,LCDR2 and LCDR3) contained within a light chain variable region (LCVR)amino acid sequence selected from the group consisting of SEQ ID NOs:10, 26, 42, 58, 74, 90, 106, 122, 138 and 154.

In one embodiment, the present invention provides an isolated antibodyor antigen-binding fragment thereof that specifically binds to GCG andneutralizes at least one activity associated with GCG, wherein theantibody or antigen-binding fragment comprises an HCVR having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 2, 18,34, 50, 66, 82, 98, 114, 130 and 146 and a LCVR having an amino acidsequence selected from the group consisting of SEQ ID NOs: 10, 26, 42,58, 74, 90, 106, 122, 138 and 154.

In one embodiment, the present invention provides an isolated antibodyor antigen-binding fragment thereof that specifically binds to GCG andneutralizes at least one activity associated with GCG, wherein theantibody or antigen-binding fragment comprises a HCVR/LCVR amino acidsequence pair selected from the group consisting of SEQ ID NOs: 2/10;18/26; 34/42; 50/58; 66/74; 82/90; 98/106; 114/122; 130/138 and 146/154.

In certain embodiments, the HCVR/LCVR amino acid sequence pair comprisesSEQ ID NOs: 18/26.

In certain embodiments, the HCVR/LCVR amino acid sequence pair comprisesSEQ ID NOs: 34/42.

In one embodiment, the present invention provides an isolated antibodyor antigen-binding fragment thereof that specifically binds to GCG andneutralizes at least one activity associated with GCG, wherein theantibody or antigen-binding fragment comprises:

(a) a HCDR1 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132 and 148;

(b) a HCDR2 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134 and 150;

(c) a HCDR3 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136 and 152;

(d) a LCDR1 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140 and 156;

(e) a LCDR2 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, and158; and

(f) a LCDR3 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144 and 160.

In one embodiment, the invention provides an antibody that specificallybinds GCG and neutralizes at least one activity associated with GCG,wherein the antibody comprises:

(a) a HCDR1 domain comprising the amino acid sequence of SEQ ID NO: 20;

(b) a HCDR2 domain comprising the amino acid sequence of SEQ ID NO: 22;

(c) a HCDR3 domain comprising the amino acid sequence of SEQ ID NO: 24;

(d) a LCDR1 domain comprising the amino acid sequence of SEQ ID NO: 28;

(e) a LCDR2 domain comprising the amino acid sequence of SEQ ID NO: 30;and

(f) a LCDR3 domain comprising the amino acid sequence of SEQ ID NO: 32.

In one embodiment, the invention provides an antibody that specificallybinds GCG and neutralizes at least one activity associated with GCG,wherein the antibody comprises:

(a) a HCDR1 domain comprising the amino acid sequence of SEQ ID NO: 36;

(b) a HCDR2 domain comprising the amino acid sequence of SEQ ID NO: 38;

(c) a HCDR3 domain comprising the amino acid sequence of SEQ ID NO: 40;

(d) a LCDR1 domain comprising the amino acid sequence of SEQ ID NO: 44;

(e) a LCDR2 domain comprising the amino acid sequence of SEQ ID NO: 46;and

(f) a LCDR3 domain comprising the amino acid sequence of SEQ ID NO: 48.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind GCG, comprising a heavy chainCDR1 (HCDR1) comprising an amino acid sequence selected from any of theHCDR1 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind GCG, comprising a heavy chainCDR2 (HCDR2) comprising an amino acid sequence selected from any of theHCDR2 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind GCG, comprising a heavy chainCDR3 (HCDR3) comprising an amino acid sequence selected from any of theHCDR3 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind GCG, comprising a light chainCDR1 (LCDR1) comprising an amino acid sequence selected from any of theLCDR1 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind GCG, comprising a light chainCDR2 (LCDR2) comprising an amino acid sequence selected from any of theLCDR2 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind GCG, comprising a light chainCDR3 (LCDR3) comprising an amino acid sequence selected from any of theLCDR3 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind GCG, comprising an HCDR3 and anLCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3amino acid sequences listed in Table 1 paired with any of the LCDR3amino acid sequences listed in Table 1. According to certainembodiments, the present invention provides antibodies, orantigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acidsequence pair contained within any of the exemplary anti-GCG antibodieslisted in Table 1. In certain embodiments, the HCDR3/LCDR3 amino acidsequence pair comprises SEQ ID NOs: 24/32.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind GCG, comprising a set of sixCDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any ofthe exemplary anti-GCG antibodies listed in Table 1. In certainembodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequenceset comprises SEQ ID NOs: 20-22-24-28-30-32. In certain embodiments, theHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set comprisesSEQ ID NOs: 36-38-40-44-46-48.

In a related embodiment, the present invention provides antibodies, orantigen-binding fragments thereof that specifically bind GCG, comprisinga set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) containedwithin an HCVR/LCVR amino acid sequence pair as defined by any of theexemplary anti-GCG antibodies listed in Table 1. For example, thepresent invention includes antibodies or antigen-binding fragmentsthereof that specifically bind GCG, comprising theHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set containedwithin an HCVR/LCVR amino acid sequence pair selected from the groupconsisting of: 18/26; 34/42; 50/58; 66/74; 82/90; 98/106; 114/122;130/138 and 146/154. Methods and techniques for identifying CDRs withinHCVR and LCVR amino acid sequences are well known in the art and can beused to identify CDRs within the specified HCVR and/or LCVR amino acidsequences disclosed herein. Exemplary conventions that can be used toidentify the boundaries of CDRs include, e.g., the Kabat definition, theChothia definition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.Sci. USA 86:9268-9272 (1989). Public databases are also available foridentifying CDR sequences within an antibody.

In one embodiment, the invention provides a fully human monoclonalantibody or antigen-binding fragment thereof that neutralizes GCGactivity, wherein the antibody or fragment thereof exhibits one or moreof the following characteristics: (i) comprises a HCVR having an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, 18,34, 50, 66, 82, 98, 114, 130 and 146; (ii) comprises a LCVR having anamino acid sequence selected from the group consisting of SEQ ID NO: 10,26, 42, 58, 74, 90, 106, 122, 138 and 154; (iii) comprises a HCDR3domain having an amino acid sequence selected from the group consistingof SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136 and 152, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; and a LCDR3 domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144 and 160, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; (iv) comprises a HCDR1 domain havingan amino acid sequence selected from the group consisting of SEQ ID NO:4, 20, 36, 52, 68, 84, 100, 116, 132 and 148, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; a HCDR2 domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54,70, 86, 102, 118, 134 and 150, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity; a LCDR1 domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108,124, 140 and 156, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;and a LCDR2 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142 and 158,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; (v)demonstrates a K_(D) of less than about 1 nM at 25° C. and less thanabout 5 nM at 37° C. as measured by surface plasmon resonance; (vi)lowers blood glucose levels by at least 10% when administered to amammal at a single dose or at multiple doses of less than about 40mg/kg; (vi) inhibits GCG activation of cells expressing the humanglucagon receptor with an IC₅₀ of less than about 1 nM.

In a second aspect, the present invention also provides nucleic acidmolecules encoding anti-GCG antibodies or portions thereof. For example,the present invention provides nucleic acid molecules encoding any ofthe HCVR amino acid sequences listed in Table 1; in certain embodimentsthe nucleic acid molecule comprises a polynucleotide sequence selectedfrom any of the HCVR nucleic acid sequences listed in Table 2, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anyof the LCVR amino acid sequences listed in Table 1. In certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCVR nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR1 amino acid sequences listed in Table 1. In certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR2 amino acid sequences listed in Table 1. In certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR3 amino acid sequences listed in Table 1. In certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR1 amino acid sequences listed in Table 1. In certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR2 amino acid sequences listed in Table 1. In certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR3 amino acid sequences listed in Table 1. In certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anHCVR, wherein the HCVR comprises a set of three CDRs (i.e.,HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequenceset is as defined by any of the exemplary anti-GCG antibodies listed inTable 1.

The present invention also provides nucleic acid molecules encoding anLCVR, wherein the LCVR comprises a set of three CDRs (i.e.,LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequenceset is as defined by any of the exemplary anti-GCG antibodies listed inTable 1.

The present invention also provides nucleic acid molecules encoding bothan HCVR and an LCVR, wherein the HCVR comprises an amino acid sequenceof any of the HCVR amino acid sequences listed in Table 1, and whereinthe LCVR comprises an amino acid sequence of any of the LCVR amino acidsequences listed in Table 1. In certain embodiments, the nucleic acidmolecule comprises a polynucleotide sequence selected from any of theHCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto, and a polynucleotide sequenceselected from any of the LCVR nucleic acid sequences listed in Table 2,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity thereto. Incertain embodiments according to this aspect of the invention, thenucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR andLCVR are both derived from the same anti-GCG antibody listed in Table 1.

The present invention also provides recombinant expression vectorscapable of expressing a polypeptide comprising a heavy or light chainvariable region of an anti-GCG antibody. For example, the presentinvention includes recombinant expression vectors comprising any of thenucleic acid molecules mentioned above, i.e., nucleic acid moleculesencoding any of the HCVR, LCVR, and/or CDR sequences as set forth inTable 1. Also included within the scope of the present invention arehost cells into which such vectors have been introduced, as well asmethods of producing the antibodies or portions thereof by culturing thehost cells under conditions permitting production of the antibodies orantibody fragments, and recovering the antibodies and antibody fragmentsso produced.

The present invention includes anti-GCG antibodies having a modifiedglycosylation pattern. In some embodiments, modification to removeundesirable glycosylation sites may be useful, or an antibody lacking afucose moiety present on the oligosaccharide chain, for example, toincrease antibody dependent cellular cytotoxicity (ADCC) function (seeShield et al. (2002) JBC 277:26733). In other applications, modificationof galactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

In a third aspect, the invention provides a pharmaceutical compositioncomprising a recombinant human antibody or fragment thereof, whichspecifically binds GCG and a pharmaceutically acceptable carrier. In arelated aspect, the invention features a composition, which is acombination of an anti-GCG antibody and a second therapeutic agent. Inone embodiment, the second therapeutic agent is any agent that isadvantageously combined with an anti-GCG antibody.

In one embodiment, the second therapeutic agent may be an agent capableof lowering blood glucose or reducing at least one symptom in a patientsuffering from a disease or condition characterized by high bloodglucose levels, such as diabetes mellitus.

In certain embodiments, the second therapeutic agent may be an agentthat helps to counteract or reduce any possible side effect(s)associated with the antibody or antigen-binding fragment of an antibodyof the invention, if such side effect(s) should occur. For example, inthe event that any of the anti-GCG antibodies increases lipid orcholesterol levels, it may be beneficial to administer a second agentthat is effective to lower lipid or cholesterol levels.

The second therapeutic agent may be a small molecule drug, aprotein/polypeptide, an antibody, a nucleic acid molecule, such as ananti-sense molecule, or a siRNA. The second therapeutic agent may besynthetic or naturally derived.

In one embodiment, the second therapeutic agent may be a glucagonreceptor antagonist, or a second glucagon antagonist, such as anotherantibody to glucagon, such as those described herein, or an antibodythat is different than the antibodies described herein. It will also beappreciated that the antibodies and pharmaceutically acceptablecompositions of the present invention can be employed in combinationtherapies, that is, the antibodies and pharmaceutically acceptablecompositions can be administered concurrently with, prior to, orsubsequent to, one or more other desired therapeutics or medicalprocedures. The particular combination of therapies (therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will also be appreciatedthat the therapies employed may achieve a desired effect for the samedisorder (for example, an antibody may be administered concurrently withanother agent used to treat the same disorder), or they may achievedifferent effects (e.g., control of any adverse effects). As usedherein, additional therapeutic agents that are normally administered totreat or prevent a particular disease, or condition, are appropriate forthe disease, or condition, being treated.

In one embodiment, the anti-GCG antibodies of the invention may be usedin combination with one or more of the following diabetes treatmentscurrently available. These include a biguanide (metformin), asulfonylurea (such as glyburide, glipizide), a peroxisomeproliferator-activated receptor (PPAR) gamma agonist (for example,pioglitazone, rosiglitazone), an alpha glucosidase inhibitor (forexample, acarbose, voglibose). Additional treatments include injectabletreatments such as a glucagon-like peptide 1 agonist or analogue (forexample, BYETTA® (exenatide), VICTOZA® (liraglutide), TANZEUM™(albiglutide), TRULICITY™ (dulaglutide), or LYXUMIA® (lixisenatide)), orwith an analogue of amylin, such as SYMLIN® (pramlintide).

In certain embodiments, the composition may include a second agentselected from the group consisting of non-sulfonylurea secretagogues,insulin, insulin analogs, including fast acting (e.g. Lispro, Aspart,Glulisine) and long acting (e.g. Detemir insulin, Degludec insulin, orGlargine insulin, exendin-4 polypeptides, beta 3 adrenoceptor agonists,PPAR agonists, a dipeptidyl peptidase IV (DPP-4) inhibitor (for example,saxagliptin (ONGLYZA®), sitaliptin (JANUVIA®), and vildagliptin(GALVUS®)), a sodium-glucose co-transporter 2 (SGLT2) inhibitor (forexample, INVOKANA™ (canagliflozin)), FORXIGA® (dapagliflozin),empagliflozin, ipragliflozin, tofogliflozin, statins andstatin-containing combinations, inhibitors of cholesterol uptake and/orbile acid re-absorption, LDL-cholesterol antagonists, cholesteryl estertransfer protein antagonists, endothelin receptor antagonists, growthhormone antagonists, insulin sensitizers, amylin mimetics or agonists,cannabinoid receptor antagonists, glucagon-like peptide-1 receptoragonists, melanocortins, melanin-concentrating hormone receptoragonists, SNRIs, a fibroblast growth factor 21 (FGF21) mimetic (See, forexample, US20110002845 and US20080261236), a fibroblast growth factorreceptor 1c (FGFR1c) agonist (See, for example, US20110150901), aninhibitor of advanced glycation endproduct formation, such as, but notlimited to, aminoguanidine, and protein tyrosine phosphatase inhibitors.

In certain embodiments, the composition may include a second agent tohelp lower lipid or cholesterol levels and may include an agent such asa 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor(for example, a statin such as atorvastatin, (LIPITOR®), fluvastatin(LESCOL®), lovastatin (MEVACOR®), pitavastatin (LIVALO®), pravastatin(PRAVACHOL®), rosuvastatin (CRESTOR®) and simvastatin (ZOCOR®) and thelike. Alternatively, the antibodies of the invention may be combinedwith an agent such as VYTORIN®, which is a preparation of a statin andanother agent, such as ezetimibe/simvastatin.

In certain embodiments, it may be beneficial to administer theantibodies of the invention in combination with any one or more of thefollowing: (1) niacin, which increases lipoprotein catabolism; (2)fibrates or amphipathic carboxylic acids, which reduce low-densitylipoprotein (LDL) level, improve high-density lipoprotein (HDL) andtriglycerides (TG) levels, and reduce the number of non-fatal heartattacks; and (3) activators of the LXR transcription factor that plays arole in cholesterol elimination such as 22-hydroxycholesterol, or fixedcombinations such as VYTORIN®) (ezetimibe plus simvastatin); a statinwith a bile resin (e.g., cholestyramine, colestipol, colesevelam), afixed combination of niacin plus a statin (e.g., niacin withlovastatin); or with other lipid lowering agents such as omega-3-fattyacid ethyl esters (for example, omacor). Furthermore, the secondtherapeutic agent can be one or more other inhibitors of glucagon or theglucagon receptor, as well as inhibitors of other molecules, such asangiopoietin-like protein 3 (ANGPTL3), angiopoietin-like protein 4(ANGPTL4), angiopoietin-like protein 5 (ANGPTL5), angiopoietin-likeprotein 6 (ANGPTL6), angiopoietin-like protein 8 (ANGPTL8), which areinvolved in lipid metabolism, in particular, cholesterol and/ortriglyceride homeostasis. Inhibitors of these molecules include smallmolecules and antibodies that specifically bind to these molecules andblock their activity.

In certain embodiments, it may be beneficial to administer the anti-GCGantibodies of the invention in combination with a nucleic acid thatinhibits the activity of hPCSK9, such as an antisense molecule, a doublestranded RNA, or a siRNA molecule. Exemplary nucleic acid molecules thatinhibit the activity of PCSK9 are described in US2011/0065644,US2011/0039914, US2008/0015162 (now U.S. Pat. No. 9,045,754) andUS2007/0173473.

In certain embodiments, it may be beneficial to administer the anti-GCGantibodies of the invention in combination with an antibody thatspecifically binds to and inhibits the activity of hPCSK9, wherein suchantibody acts to lower lipid or cholesterol levels. Exemplaryanti-hPCSK9 antibodies are described in US2010/0166768 (now U.S. Pat.No. 8,062,640). The isolated antibody that specifically binds to humanPCSK9, or an antigen-binding fragment thereof, may be administered at adose ranging from about 0.01 mg/kg to about 30 mg/kg. It may beadministered as a single dose or as multiple doses. The anti-hPCSK9antibody may be administered concurrently with the anti-GCG antibody, orit may be administered prior to, or after the anti-GCG antibody.

In a fourth aspect, the invention features a human anti-hGCGR antibodyor antigen-binding fragment of an antibody comprising a HCVR encoded bynucleotide sequence segments derived from V_(H), D_(H) and J_(H)germline sequences, and a LCVR encoded by nucleotide sequence segmentsderived from V_(K) and J_(K) germline sequences, with combinations asshown in Table 3.

In a fifth aspect, the invention features methods for inhibiting GCGactivity using an anti-GCG antibody or antigen-binding portion of theantibody of the invention, wherein the therapeutic methods compriseadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising an antibody or antigen-binding fragment thereof.The antibodies of the invention may be used to treat any condition ordisorder, which is improved, ameliorated, inhibited or prevented byremoval, inhibition or reduction of GCG activity.

In one embodiment, the antibodies may be used to prevent the onset of adisease or disorder characterized in part by elevated blood glucoselevels, or to prevent the likelihood of developing such disease ordisorder, or to mitigate the severity of the disease or disorder, or atleast one symptom associated with the disease or disorder. It isenvisioned that the antibodies of the invention may be used alone, or asadjunct therapy with other agents or methods known to be standard carefor treating patients suffering from diseases or conditionscharacterized in part by elevated blood glucose or ketone levels, suchas, but not limited to, diabetes. Such standard therapy may includefluid administration, or administration of any other pharmaceuticalagents useful for lowering blood glucose, ketones, or lipids, or forweight reduction.

The anti-GCG antibodies of the invention may function to block theinteraction between glucagon and its receptor, thereby inhibiting theglucose elevating effects of glucagon. The use of glucagon antagonists,such as the antibodies described herein, may be an effective means ofachieving normal levels of glucose, thereby ameliorating, or preventingone or more symptoms of, or long term complications associated with, forexample, diabetes. The use of glucagon antagonists, such as theantibodies described herein, may also be an effective means of achievingnormal levels of glucose in non-diabetic patients, who experiencehyperglycemia as a result of conditions or disorders not related todiabetes, such as perioperative hyperglycemia (hyperglycemia observed inpatients just prior to surgery, or after surgery). In certainembodiments, methods of lowering blood glucose levels or ketone levelsin diabetic ketoacidosis are envisioned using the antibodies of theinvention. In certain embodiments, methods of treating patients toachieve a reduction in body weight, or to prevent weight gain, or tomaintain a normal body weight, are also envisioned using the antibodiesof the invention.

The antibodies of the present invention may be useful for amelioratingconditions such as, for example, impaired glucose tolerance, obesity, orfor preventing weight gain, or for treating diabetic conditions, or forpreventing or reducing the severity of any one or more of the long-termcomplications associated with diabetes, such as nephropathy, neuropathy,retinopathy, cataracts, stroke, atherosclerosis, impaired wound healingand other complications associated with diabetes, known to those skilledin the art.

In one embodiment, the antibodies of the invention may be used fortreating stress hyperglycemia, or for preventing the likelihood of onsetof stress hyperglycemia in a patient, (also referred to as“stress-induced hyperglycemia”), the method comprising administering toa patient a therapeutically effective amount of a composition comprisinga glucagon antibody of the invention, wherein the patient exhibitselevated levels of blood glucose caused, or exacerbated by, one or morestress-inducing stimulus or one or more glucose elevating stimulus. Inone embodiment, the patient is identified on the basis of having a bloodglucose level greater than about 140 mg/dL.

In one embodiment, the stress-inducing stimulus or the glucose elevatingstimulus is selected from the group consisting of: pre-existing type 1or type 2 diabetes; hypertonic dehydration; infusion of catecholaminepressors; glucocorticoid therapy; obesity; aging; excessive dextroseadministration; parenteral nutrition, enteral nutrition, pancreatitis;sepsis; stroke; traumatic head injury; hypothermia; hypoxemia; uremia;cirrhosis; anesthesia; pre-operative or post-operative hospital stays(pen-operative hyperglycemia); admission to an emergency room, a traumacenter, or an intensive care unit; prolonged hospital stays; surgicalprocedures; an infection; or a chronic illness.

Other conditions or disorders treatable by the therapeutic methods ofthe invention include diabetic ketoacidosis, perioperativehyperglycemia, hyperglycemia in the intensive care unit patient,hyperosmolar hyperglycemia syndrome, hyperglycemia in burn patients,hyperglycemia is patients suffering from a cardiac condition,hyperinsulinemia, the metabolic syndrome, insulin resistance syndrome,impaired fasting glucose, or hyperglycemia associated withhypercholesterolemia, hypertriglyceridemia, hyperlipidemia, and generaldyslipidemias.

The therapeutic methods according to this aspect of the inventioncomprise administering a therapeutically effective amount of apharmaceutical composition comprising an antibody or antigen-bindingfragment of an antibody of the invention to a subject in need thereof.The disorder treated is any disease or condition which is improved,ameliorated, inhibited or prevented by targeting GCG.

The antibodies may be useful for treating patients with inoperableglucagonoma (pancreatic endocrine tumor with or without necrolyticmigratory erythema and hyperglycemia).

The antibodies of the invention may be used as short-term therapy in anacute setting, or they may be envisioned for long-term use as chronictherapy.

Other embodiments will become apparent from a review of the ensuingdetailed description.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Definitions

“Glucagon” or, “GCG,” and the like, as used herein, refers to humanglucagon (unless glucagon from another species is specifically noted),comprising the amino acid sequence as set forth in SEQ ID NO: 161. Seealso amino acid residues 53-81 of accession number NP_002045.1.

All references to proteins, polypeptides and protein fragments hereinare intended to refer to the human version of the respective protein,polypeptide or protein fragment unless explicitly specified as beingfrom a non-human species. Thus, the expression “GCG” means human GCGunless specified as being from a non-human species, e.g., “mouse GCG,”“monkey GCG,” etc.

The term “human proprotein convertase subtilisin/kexin type 9” or“hPCSK9”, as used herein, refers to hPCSK9 encoded by the nucleic acidsequence shown in SEQ ID NO:162 and having the amino acid sequence ofSEQ ID NO:163, or a biologically active fragment thereof.

The specific embodiments, antibody or antibody fragments of theinvention may be conjugated to a therapeutic moiety (“immunoconjugate”),such as a second GCG antagonist, or to biguanide (metformin), asulfonylurea (such as glyburide, glipizide), a PPAR gamma agonist (suchas pioglitazone, or rosiglitazone), an alpha glucosidase inhibitor (suchas acarbose, or voglibose), BYETTA® (glucagon-like peptide 1), SYMLIN®(pramlintide), or any other therapeutic moiety useful for treating adisease or condition caused in part by unwanted glucagon activity.

As used herein, the expression “anti-GCG antibody” includes bothmonovalent antibodies with a single specificity, as well as bispecificantibodies comprising a first arm that binds GCG and a second arm thatbinds a second (target) antigen, wherein the anti-GCG arm comprises anyof the HCVR/LCVR or CDR sequences as set forth in Table 1 herein.

The term “antibody”, as used herein, means any antigen-binding moleculeor molecular complex comprising at least one complementarity determiningregion (CDR) that specifically binds to or interacts with a particularantigen (e.g., GCG). The term “antibody” includes immunoglobulinmolecules comprising four polypeptide chains, two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, as well asmultimers thereof (e.g., IgM). Each heavy chain comprises a heavy chainvariable region (abbreviated herein as HCVR or V_(H)) and a heavy chainconstant region. The heavy chain constant region comprises threedomains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a lightchain variable region (abbreviated herein as LCVR or V_(L)) and a lightchain constant region. The light chain constant region comprises onedomain (C_(L)1). The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FR). Each V_(H) and V_(L) is composed of threeCDRs and four FRs, arranged from amino-terminus to carboxy-terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In differentembodiments of the invention, the FRs of the anti-GCG antibody (orantigen-binding portion thereof) may be identical to the human germlinesequences, or may be naturally or artificially modified. An amino acidconsensus sequence may be defined based on a side-by-side analysis oftwo or more CDRs.

The term “antibody”, as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDR,which is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The term “human antibody”, as used herein, is intended to includenon-naturally occurring human antibodies. The term includes antibodiesthat are recombinantly produced in a non-human mammal, or in cells of anon-human mammal. The term is not intended to include antibodiesisolated from or generated in a human subject.

The antibodies of the invention may, in some embodiments, be recombinanthuman antibodies. The term “recombinant human antibody”, as used herein,is intended to include all human antibodies that are prepared,expressed, created or isolated by recombinant means, such as antibodiesexpressed using a recombinant expression vector transfected into a hostcell (described further below), antibodies isolated from a recombinant,combinatorial human antibody library (described further below),antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. In certainembodiments, such recombinant human antibodies are subjected to in vitromutagenesis (or, when an animal transgenic for human Ig sequences isused, in vivo somatic mutagenesis) and thus the amino acid sequences ofthe V_(H) and V_(L) regions of the recombinant antibodies are sequencesthat, while related to human germline V_(H) and V_(L) sequences, may notnaturally exist within the human antibody germline repertoire in vivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

The term “specifically binds”, or “binds specifically to”, or the like,means that an antibody or antigen-binding fragment thereof forms acomplex with an antigen that is relatively stable under physiologicconditions. Specific binding can be characterized by an equilibriumdissociation constant of at least about 1×10⁻⁶ M or less (e.g., asmaller K_(D) denotes a tighter binding). Methods for determiningwhether two molecules specifically bind are well known in the art andinclude, for example, equilibrium dialysis, surface plasmon resonance,and the like. As described herein, antibodies have been identified bysurface plasmon resonance, e.g., BIACORE™, which bind specifically toGCG. Moreover, multispecific antibodies that bind to GCG protein and oneor more additional antigens or a bi-specific that binds to two differentregions of GCG are nonetheless considered antibodies that “specificallybind”, as used herein.

The antibodies of the invention may be isolated antibodies. An “isolatedantibody,” as used herein, means an antibody that has been identifiedand separated and/or recovered from at least one component of itsnatural environment. For example, an antibody that has been separated orremoved from at least one component of an organism, or from a tissue orcell in which the antibody naturally exists or is naturally produced, isan “isolated antibody” for purposes of the present invention. Anisolated antibody also includes an antibody in situ within a recombinantcell. Isolated antibodies are antibodies that have been subjected to atleast one purification or isolation step. According to certainembodiments, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The anti-GCG antibodies disclosed herein may comprise one or more aminoacid substitutions, insertions and/or deletions in the framework and/orCDR regions of the heavy and light chain variable domains. Suchmutations can be readily ascertained by comparing the amino acidsequences disclosed herein to sequences available from, for example,public antibody sequence databases. Once obtained, antibodies andantigen-binding fragments that contain one or more mutations can beeasily tested for one or more desired property such as, improved bindingspecificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-GCG antibodies comprisingvariants of any of the HCVR, LCVR, and/or CDR amino acid sequencesdisclosed herein having one or more conservative substitutions. Forexample, the present invention includes anti-GCG antibodies having HCVR,LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 orfewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences set forth in Table 1 herein.

A “blocking antibody” or a “neutralizing antibody”, as used herein (oran “antibody that neutralizes GCG activity”), is intended to refer to anantibody whose binding to glucagon prevents or blocks its binding to theglucagon receptor, which results in inhibition of at least onebiological activity of GCG. For example, an antibody of the inventionmay aid in preventing the increase in blood glucose levels associatedwith elevation of glucagon levels. Alternatively, an antibody of theinvention may demonstrate the ability to block cAMP production inresponse to glucagon. This inhibition of the biological activity of GCGcan be assessed by measuring one or more indicators of GCG biologicalactivity by one or more of several standard in vitro or in vivo assaysknown in the art.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-timebiomolecular interactions by detection of alterations in proteinconcentrations within a biosensor matrix, for example using the BIACORE™system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24: 307-331, herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443-1445, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

By the phrase “therapeutically effective amount” is meant an amount thatproduces the desired effect for which it is administered. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see, forexample, Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

The term “blood glucose level”, or “level of blood glucose” shall meanblood glucose concentration. In certain embodiments, a blood glucoselevel is a plasma glucose level. Plasma glucose may be determined inaccordance with Etgen et al., (Metabolism 2000; 49(5): 684-688) orcalculated from a conversion of whole blood glucose concentration inaccordance with D'Orazio et al., (Clin. Chem. Lab. Med. 2006; 44(12):1486-1490).

“Normal glucose levels” refers to mean plasma glucose values in humansof less than about 100 mg/dL for fasting levels, and less than 110-120mg/dL for 2-hour postprandial levels or 125 mg/dL for a random glucose.Plasma glucose may be determined in accordance with Etgen et al.,(Metabolism 2000; 49(5): 684-688) or calculated from a conversion ofwhole blood glucose concentration in accordance with D'Orazio et al.,(Clin. Chem. Lab. Med. 2006; 44(12): 1486-1490). In certain embodimentsof the invention, the anti-GCG antibodies may be useful to lower bloodglucose levels to within the normal range.

The term “elevated blood glucose level” or “elevated levels of bloodglucose” shall mean an elevated blood glucose level such as that foundin a subject demonstrating clinically inappropriate basal andpostprandial hyperglycemia or such as that found in a subject in oralglucose tolerance test (oGTT), with “elevated levels of blood glucose”being greater than about 100 mg/dL when tested under fasting conditions,and greater than about 200 mg/dL when tested at 1 hour.

The term “stress hyperglycemia”, which is used interchangeably with“stress-induced hyperglycemia”, refers to a condition whereby a patientsuffers from a transient increase in blood glucose (>140 mg/dL) that istemporally linked to the stress of an acute injury or illness. Stresshyperglycemia can occur in patients with or without a history ofdiabetes. The cause is thought to be directly related to the stress ofthe underlying medical illness, anesthesia, surgery, or trauma.

Stress hyperglycemia is the result of, or may be exacerbated by, any oneor more of the following risk factors, conditions or therapies:pre-existing type 1 or type 2 diabetes; hypertonic dehydration; infusionof catecholamine pressors; glucocorticoid therapy; obesity; aging;excessive dextrose administration; parenteral nutrition, enteralnutrition, pancreatitis; sepsis; stroke; traumatic head injury;hypothermia; hypoxemia; uremia; cirrhosis; anesthesia; pre-operative orpost-operative hospital stays (peri-operative hyperglycemia); admissionto an emergency room, a trauma center, or an intensive care unit;prolonged hospital stays; surgical procedures; an infection; or aworsening chronic illness.

The term “critically ill”, as used herein, generally refers to a patientsuffering from a disease, disorder, injury, surgical procedure, or othercondition who requires treatment or monitoring in a critical care unit,or an intensive care unit of a hospital. In its broadest sense, the terma “critically ill” patient, as used herein refers to a patient who hassustained or is at risk of sustaining acutely life-threatening single ormultiple organ system failure. A critically ill patient may be a“diabetic patient”, e.g. a patient having been diagnosed as havingdiabetes using standard tests known to those skilled in the art; or a“non-diabetic patient”, e.g. a patient who has been diagnosed as nothaving diabetes using standard methods known to those skilled in theart.

The term “not-critically ill”, or “non-critically ill” refers to ahospitalized patient suffering from a disease, disorder, or conditionthat does not require treatment or monitoring in a critical care unit,or intensive care unit of a hospital. In its broadest sense, the term a“not-critically ill” patient, as used herein refers to a patient otherthan one who has sustained or is at risk of sustaining acutelylife-threatening single or multiple organ system failure due to disease,injury, surgical procedure, or other condition.

The term “Intensive Care Unit” (herein designated ICU), as used hereinrefers to the part of a hospital where critically ill patients aretreated. This might vary from country to country and even from hospitalto hospital and this part of the hospital may not necessary, officially,bear the name “Intensive Care Unit” or a translation or derivationthereof. The term “Intensive Care Unit” also covers any health care unitthat treats patients with life-threatening conditions requiringconstant, close monitoring and support from equipment and medication inorder to maintain normal bodily functions.

The term “treating” or “treatment”, as used herein, refers to anapproach for obtaining beneficial or desired clinical results. Forpurposes of this invention, beneficial or desired clinical resultsinclude, but are not limited to, one or more of the following:improvement in blood glucose to within about 80-180 mg/dL, or to withinabout 80-140 mg/dL, or an improvement in any one or more conditions,diseases, or symptoms associated with, or resulting from, elevatedlevels of blood glucose, including, but not limited to susceptibility toinfections, organ failure, disability after stroke, polyneuropathy,arrhythmia, or mortality in patients. In addition, “treating” with aglucagon antagonist/antibody of the invention may result in a beneficialor desired clinical result which may include an improvement in bloodglucose level to within about 80-180 mg/dL, or to within about 80-140mg/dL, in any condition or disease resulting from exposure to any one ormore stress-inducing stimulus or glucose elevating stimulus selectedfrom the group consisting of: pre-existing type 1 or type 2 diabetes;infusion of catecholamine pressors; parenteral nutrition; enteralnutrition; glucocorticoid therapy; obesity; aging; excessive dextroseadministration; pancreatitis; sepsis; stroke; traumatic head injury;hypothermia; hypoxemia; uremia; cirrhosis; anesthesia; pre-operative orpost-operative hospital stays (pen-operative hyperglycemia); admissionto an emergency room, a trauma center, or an intensive care unit;prolonged hospital stays; surgical procedures; an infection; and achronic illness. “Treating” with a glucagon antagonist/antibody of theinvention may also lead to prevention of the onset of stresshyperglycemia, or to prevention of the likelihood of onset of stresshyperglycemia.

A “stress-inducing stimulus”, which is used interchangeably with a“glucose-elevating stimulus”, refers to an event that promotes theelevation of blood glucose to above-normal levels. Examples of a“stress-inducing stimulus”, or a “glucose-elevating stimulus” includeany one or more of the following: pre-existing type 1 or type 2diabetes; hypertonic dehydration; infusion of catecholamine pressors;parenteral nutrition; enteral nutrition; glucocorticoid therapy;obesity; aging; excessive dextrose administration; pancreatitis; sepsis;stroke; traumatic head injury; hypothermia; hypoxemia; uremia;cirrhosis; anesthesia; pre-operative or post-operative hospital stays(pen-operative hyperglycemia); admission to an emergency room, a traumacenter, or an intensive care unit; prolonged hospital stays; surgicalprocedures; an infection; and a chronic illness.

The term “insulin”, as used herein refers to insulin from any speciessuch as human insulin, porcine insulin, bovine insulin and saltsthereof, such as zinc salts.

pH-Dependent Binding

The present invention includes anti-GCG antibodies with pH-dependentbinding characteristics. For example, an anti-GCG antibody of thepresent invention may exhibit reduced binding to GCG at acidic pH ascompared to neutral pH. Alternatively, anti-GCG antibodies of theinvention may exhibit enhanced binding to GCG at acidic pH as comparedto neutral pH. The expression “acidic pH” includes pH values less thanabout 6.2, e.g., about 6.0, 5.95, 5, 9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6,5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, orless. As used herein, the expression “neutral pH” means a pH of about7.0 to about 7.4. The expression “neutral pH” includes pH values ofabout 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

In certain instances, “reduced binding to GCG at acidic pH as comparedto neutral pH” is expressed in terms of a ratio of the K_(D) value ofthe antibody binding to GCG at acidic pH to the K_(D) value of theantibody binding to GCG at neutral pH (or vice versa). For example, anantibody or antigen-binding fragment thereof may be regarded asexhibiting “reduced binding to GCG at acidic pH as compared to neutralpH” for purposes of the present invention if the antibody orantigen-binding fragment thereof exhibits an acidic/neutral K_(D) ratioof about 3.0 or greater. In certain exemplary embodiments, theacidic/neutral K_(D) ratio for an antibody or antigen-binding fragmentof the present invention can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5,13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0,100.0 or greater.

Antibodies with pH-dependent binding characteristics may be obtained,e.g., by screening a population of antibodies for reduced (or enhanced)binding to a particular antigen at acidic pH as compared to neutral pH.Additionally, modifications of the antigen-binding domain at the aminoacid level may yield antibodies with pH-dependent characteristics. Forexample, by substituting one or more amino acids of an antigen-bindingdomain (e.g., within a CDR) with a histidine residue, an antibody withreduced antigen-binding at acidic pH relative to neutral pH may beobtained.

Anti-GCG Antibodies Comprising Fc Variants

According to certain embodiments of the present invention, anti-GCGantibodies are provided comprising an Fc domain comprising one or moremutations which enhance or diminish antibody binding to the FcRnreceptor, e.g., at acidic pH as compared to neutral pH. For example, thepresent invention includes anti-GCG antibodies comprising a mutation inthe C_(H)2 or a C_(H)3 region of the Fc domain, wherein the mutation(s)increases the affinity of the Fc domain to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Suchmutations may result in an increase in serum half-life of the antibodywhen administered to an animal. Non-limiting examples of such Fcmodifications include, e.g., a modification at position 250 (e.g., E orQ); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., Sor T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., A, W, H, F or Y[N434A, N434W, N434H, N434F or N434Y]); or a modification at position250 and/or 428; or a modification at position 307 or 308 (e.g., 308F,V308F), and 434. In one embodiment, the modification comprises a 428L(e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g.,V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T,and 256E) modification; a 250Q and 428L modification (e.g., T250Q andM428L); and a 307 and/or 308 modification (e.g., 308F or 308P). In yetanother embodiment, the modification comprises a 265A (e.g., D265A)and/or a 297A (e.g., N297A) modification.

For example, the present invention includes anti-GCG antibodiescomprising an Fc domain comprising one or more pairs or groups ofmutations selected from the group consisting of: 250Q and 248L (e.g.,T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E);428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I andQ311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g.,D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A);and 433K and 434F (e.g., H433K and N434F). All possible combinations ofthe foregoing Fc domain mutations, and other mutations within theantibody variable domains disclosed herein, are contemplated within thescope of the present invention.

The present invention also includes anti-GCG antibodies comprising achimeric heavy chain constant (C_(H)) region, wherein the chimeric C_(H)region comprises segments derived from the C_(H) regions of more thanone immunoglobulin isotype. For example, the antibodies of the inventionmay comprise a chimeric C_(H) region comprising part or all of a C_(H)2domain derived from a human IgG1, human IgG2 or human IgG4 molecule,combined with part or all of a C_(H)3 domain derived from a human IgG1,human IgG2 or human IgG4 molecule. According to certain embodiments, theantibodies of the invention comprise a chimeric C_(H) region having achimeric hinge region. For example, a chimeric hinge may comprise an“upper hinge” amino acid sequence (amino acid residues from positions216 to 227 according to EU numbering) derived from a human IgG1, a humanIgG2 or a human IgG4 hinge region, combined with a “lower hinge”sequence (amino acid residues from positions 228 to 236 according to EUnumbering) derived from a human IgG1, a human IgG2 or a human IgG4 hingeregion. According to certain embodiments, the chimeric hinge regioncomprises amino acid residues derived from a human IgG1 or a human IgG4upper hinge and amino acid residues derived from a human IgG2 lowerhinge. An antibody comprising a chimeric C_(H) region as describedherein may, in certain embodiments, exhibit modified Fc effectorfunctions without adversely affecting the therapeutic or pharmacokineticproperties of the antibody. (See, e.g., U.S. Provisional Appl. No.61/759,578, filed Feb. 1, 2013, the disclosure of which is herebyincorporated by reference in its entirety).

Biological Characteristics of the Antibodies

The present invention includes antibodies and antigen-binding fragmentsthereof that bind GCG with high affinity. For example, the presentinvention includes anti-GCG antibodies that bind GCG with a K_(D) ofless than about 5.0 nM as measured by surface plasmon resonance at 25°C. or 37° C., e.g., using an assay format as defined in Example 3herein, or a substantially similar assay. According to certainembodiments, anti-GCG antibodies are provided that bind GCG at 37° C.with a K_(D) of less than about 5 nM, less than about 3 nM, less thanabout 2 nM, less than about 1 nM, less than about 900 pM, less thanabout 800 pM, less than about 700 pM, less than about 600 pM, less thanabout 500 pM, less than about 400 pM, or less than about 300 pM, asmeasured by surface plasmon resonance, e.g., using an assay format asdefined in Example 3 herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind GCG with a dissociative half-life (t ½) ofgreater than about 0.5 minutes as measured by surface plasmon resonanceat 25° C. or 37° C., e.g., using an assay format as defined in Example 3herein, or a substantially similar assay. According to certainembodiments, anti-GCG antibodies are provided that bind GCG at 37° C.with a t ½ of greater than or equal to about 0.5 minutes, greater thanabout 6 minutes, greater than about 8 minutes, greater than about 10minutes, greater than about 12 minutes, greater than about 14 minutes,greater than about 16 minutes, greater than about 18 minutes, greaterthan about 20 minutes, greater than about 30 minutes, greater than about40 minutes, or longer, as measured by surface plasmon resonance, e.g.,using an assay format as defined in Example 3 herein, or a substantiallysimilar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind GCG binding to and activation of cellsexpressing the glucagon receptor (GCGR). For example, the presentinvention includes anti-GCG antibodies that block binding of glucagon tocells that express the glucagon receptor, with an EC₅₀ of less thanabout 60 pM as measured using GCG bioassay format as defined in Example4 herein, or a substantially similar assay. According to certainembodiments, anti-GCG antibodies are provided that block activation incells expressing GCGR, with an EC₅₀ of less than about 50 nM, less thanabout 5.0 nM, less than about 1.0 nM, less than about 900 pM, less thanabout 800 pM, less than about 600 pM, less than about 400 pM, less thanabout 200 pM, less than about 100 pM, less than about 80 pM, less thanabout 60 pM, less than about 40 pM, less than about 20 pM as measuredusing a bioassay format as defined in Example 4 herein, or asubstantially similar assay.

The antibodies of the present invention may possess one or more of theaforementioned biological characteristics, or any combination thereof.The foregoing list of biological characteristics of the antibodies ofthe invention is not intended to be exhaustive. Other biologicalcharacteristics of the antibodies of the present invention will beevident to a person of ordinary skill in the art from a review of thepresent disclosure including the working Examples herein.

Epitope Mapping and Related Technologies

The epitope to which the antibodies of the present invention bind mayconsist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acidsof a GCG protein. Alternatively, the epitope may consist of a pluralityof non-contiguous amino acids (or amino acid sequences) of GCG.

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,e.g., routine cross-blocking assay such as that described Antibodies,Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY),alanine scanning mutational analysis, peptide blots analysis (Reineke,2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Inaddition, methods such as epitope excision, epitope extraction andchemical modification of antigens can be employed (Tomer, 2000, ProteinScience 9:487-496). Another method that can be used to identify theamino acids within a polypeptide with which an antibody interacts ishydrogen/deuterium exchange detected by mass spectrometry. In generalterms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water to allow hydrogen-deuterium exchange tooccur at all residues except for the residues protected by the antibody(which remain deuterium-labeled). After dissociation of the antibody,the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

The present invention further includes anti-GCG antibodies that bind tothe same epitope as any of the specific exemplary antibodies describedherein (e.g. antibodies comprising any of the amino acid sequences asset forth in Table 1 herein). Likewise, the present invention alsoincludes anti-GCG antibodies that compete for binding to GCG with any ofthe specific exemplary antibodies described herein (e.g. antibodiescomprising any of the amino acid sequences as set forth in Table 1herein).

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-GCG antibody by usingroutine methods known in the art and exemplified herein. For example, todetermine if a test antibody binds to the same epitope as a referenceanti-GCG antibody of the invention, the reference antibody is allowed tobind to a GCG protein. Next, the ability of a test antibody to bind tothe GCG molecule is assessed. If the test antibody is able to bind toGCG following saturation binding with the reference anti-GCG antibody,it can be concluded that the test antibody binds to a different epitopethan the reference anti-GCG antibody. On the other hand, if the testantibody is not able to bind to the GCG molecule following saturationbinding with the reference anti-GCG antibody, then the test antibody maybind to the same epitope as the epitope bound by the reference anti-GCGantibody of the invention. Additional routine experimentation (e.g.,peptide mutation and binding analyses) can then be carried out toconfirm whether the observed lack of binding of the test antibody is infact due to binding to the same epitope as the reference antibody or ifsteric blocking (or another phenomenon) is responsible for the lack ofobserved binding. Experiments of this sort can be performed using ELISA,RIA, Biacore, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art. In accordance with certainembodiments of the present invention, two antibodies bind to the same(or overlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excessof one antibody inhibits binding of the other by at least 50% butpreferably 75%, 90% or even 99% as measured in a competitive bindingassay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).Alternatively, two antibodies are deemed to bind to the same epitope ifessentially all amino acid mutations in the antigen that reduce oreliminate binding of one antibody reduce or eliminate binding of theother. Two antibodies are deemed to have “overlapping epitopes” if onlya subset of the amino acid mutations that reduce or eliminate binding ofone antibody reduce or eliminate binding of the other.

To determine if an antibody competes for binding (or cross-competes forbinding) with a reference anti-GCG antibody, the above-described bindingmethodology is performed in two orientations: In a first orientation,the reference antibody is allowed to bind to a GCG protein undersaturating conditions followed by assessment of binding of the testantibody to the GCG molecule. In a second orientation, the test antibodyis allowed to bind to a GCG molecule under saturating conditionsfollowed by assessment of binding of the reference antibody to the GCGmolecule. If, in both orientations, only the first (saturating) antibodyis capable of binding to the GCG molecule, then it is concluded that thetest antibody and the reference antibody compete for binding to GCG. Aswill be appreciated by a person of ordinary skill in the art, anantibody that competes for binding with a reference antibody may notnecessarily bind to the same epitope as the reference antibody, but maysterically block binding of the reference antibody by binding anoverlapping or adjacent epitope.

Preparation of Human Antibodies

The anti-GCG antibodies of the present invention can be fully human(non-naturally occurring) antibodies. Methods for generating monoclonalantibodies, including fully human monoclonal antibodies are known in theart. Any such known methods can be used in the context of the presentinvention to make human antibodies that specifically bind to human GCG.

Using VELOCIMMUNE® technology (see, for example, U.S. Pat. No.6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other knownmethod for generating monoclonal antibodies, high affinity chimericantibodies to an antigen are initially isolated having a human variableregion and a mouse constant region. The VELOCIMMUNE® technology involvesgeneration of a transgenic mouse having a genome comprising human heavyand light chain variable regions operably linked to endogenous mouseconstant region loci such that the mouse produces an antibody comprisinga human variable region and a mouse constant region in response toantigenic stimulation. The DNA encoding the variable regions of theheavy and light chains of the antibody are isolated and operably linkedto DNA encoding the human heavy and light chain constant regions. TheDNA is then expressed in a cell capable of expressing the fully humanantibody.

Generally, a VELOCIMMUNE® mouse is challenged with the antigen ofinterest, and lymphatic cells (such as B-cells) are recovered from themice that express antibodies. The lymphatic cells may be fused with amyeloma cell line to prepare immortal hybridoma cell lines, and suchhybridoma cell lines are screened and selected to identify hybridomacell lines that produce antibodies specific to the antigen of interest.DNA encoding the variable regions of the heavy chain and light chain maybe isolated and linked to desirable isotypic constant regions of theheavy chain and light chain. Such an antibody protein may be produced ina cell, such as a CHO cell. Alternatively, DNA encoding theantigen-specific chimeric antibodies or the variable domains of thelight and heavy chains may be isolated directly from antigen-specificlymphocytes.

As described in the experimental section below, the high affinitychimeric antibodies, which are isolated having a human variable regionand a mouse constant region, are characterized and selected fordesirable characteristics, including affinity, selectivity, epitope,etc. The mouse constant regions are then replaced with a desired humanconstant region to generate the fully human antibody of the invention,for example wild-type or modified IgG1 or IgG4. While the constantregion selected may vary according to specific use, high affinityantigen-binding and target specificity characteristics reside in thevariable region.

In general, the antibodies of the instant invention possess very highaffinities, typically possessing K_(D) of from about 10⁻¹² through about10⁻⁹ M, when measured by binding to antigen either immobilized on solidphase or in solution phase.

Bioequivalents

The anti-GCG antibodies and antibody fragments of the present inventionencompass proteins having amino acid sequences that vary from those ofthe described antibodies but that retain the ability to bind human GCG.Such variant antibodies and antibody fragments comprise one or moreadditions, deletions, or substitutions of amino acids when compared toparent sequence, but exhibit biological activity that is essentiallyequivalent to that of the described antibodies. Likewise, the anti-GCGantibody-encoding DNA sequences of the present invention encompasssequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an anti-GCG antibody or antibody fragment that isessentially bioequivalent to an anti-GCG antibody or antibody fragmentof the invention. Examples of such variant amino acid and DNA sequencesare discussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-GCG antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include anti-GCG antibody variantscomprising amino acid changes, which modify the glycosylationcharacteristics of the antibodies, e.g., mutations which eliminate orremove glycosylation.

Multispecific Antibodies

The antibodies of the present invention may be monospecific ormultispecific (e.g., bispecific). Multispecific antibodies may bespecific for different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The anti-GCG antibodies of the presentinvention can be linked to or co-expressed with another functionalmolecule, e.g., another peptide or protein. For example, an antibody orfragment thereof can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmentto produce a bi-specific or a multispecific antibody with a secondbinding specificity.

The present invention includes bispecific antibodies wherein one arm ofan immunoglobulin binds human GCG, and the other arm of theimmunoglobulin is specific for a second antigen. The GCG-binding arm cancomprise any of the HCVR/LCVR or CDR amino acid sequences as set forthin Table 1 herein.

An exemplary bispecific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bispecific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bispecificantibody format described above are contemplated within the scope of thepresent invention.

Other exemplary bispecific formats that can be used in the context ofthe present invention include, without limitation, e.g., scFv-based ordiabody bispecific formats, IgG-scFv fusions, dual variable domain(DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., commonlight chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab (DAF)-IgG, andMab² bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11,and references cited therein, for a review of the foregoing formats).Bispecific antibodies can also be constructed using peptide/nucleic acidconjugation, e.g., wherein unnatural amino acids with orthogonalchemical reactivity are used to generate site-specificantibody-oligonucleotide conjugates which then self-assemble intomultimeric complexes with defined composition, valency and geometry.(See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Therapeutic Formulation and Administration

The invention provides pharmaceutical compositions comprising theanti-GCG antibodies or antigen-binding fragments thereof of the presentinvention. The pharmaceutical compositions of the invention areformulated with suitable carriers, excipients, and other agents thatprovide improved transfer, delivery, tolerance, and the like. Amultitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. These formulationsinclude, for example, powders, pastes, ointments, jellies, waxes, oils,lipids, lipid (cationic or anionic) containing vesicles (such asLIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA conjugates,anhydrous absorption pastes, oil-in-water and water-in-oil emulsions,emulsions carbowax (polyethylene glycols of various molecular weights),semi-solid gels, and semi-solid mixtures containing carbowax. See alsoPowell et al. “Compendium of excipients for parenteral formulations” PDA(1998) J Pharm Sci Technol 52:238-311.

The dose of antibody administered to a patient may vary depending uponthe age and the size of the patient, target disease, conditions, routeof administration, and the like. The preferred dose is typicallycalculated according to body weight or body surface area. In an adultpatient, it may be advantageous to intravenously administer the antibodyof the present invention normally at a single dose of about 0.01 toabout 20 mg/kg body weight, more preferably about 0.02 to about 7, about0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Dependingon the severity of the condition, the frequency and the duration of thetreatment can be adjusted. Effective dosages and schedules foradministering anti-GCG antibodies may be determined empirically; forexample, patient progress can be monitored by periodic assessment, andthe dose adjusted accordingly. Moreover, interspecies scaling of dosagescan be performed using well-known methods in the art (e.g., Mordenti etal., 1991, Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L. P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla. In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Immunoconjugates

The invention encompasses a human anti-GCG monoclonal antibodyconjugated to a therapeutic moiety (“immunoconjugate”), such as an agentthat is capable of reducing blood glucose levels, or a radioisotope, ora chemotherapeutic agent. The type of therapeutic moiety that may beconjugated to the anti-GCG antibody will take into account the conditionto be treated and the desired therapeutic effect to be achieved. Forexample, for treating diabetes, or any other condition whereby it isdesirable to lower blood glucose, and/or to maintain normal bloodglucose levels, an agent such as biguanide (e.g. metformin), asulfonylurea (e.g. glyburide, glipizide), a PPAR gamma agonist (e.g.pioglitazone, rosiglitazone); an alpha glucosidase inhibitor (e.g.acarbose, voglibose), an inhibitor of advanced glycation endproductformation (e.g. aminoguanidine), or a second GCG inhibitor may beconjugated to the GCG antibody. Alternatively, if the desiredtherapeutic effect is to treat the sequelae or symptoms associated withdiabetes, or any other condition resulting from high, or uncontrolledblood glucose levels, it may be advantageous to conjugate an agentappropriate to treat the sequelae or symptoms of the condition Examplesof suitable agents for forming immunoconjugates are known in the art,see for example, WO 05/103081.

Therapeutic Uses of the Antibodies

Due to their interaction with glucagon, the present antibodies areuseful for lowering blood glucose levels and also for the treatment of awide range of conditions and disorders in which blocking the interactionof glucagon with its receptor is beneficial. These disorders andconditions may be selected from any glucagon related metabolic disorder,which involves glucagon receptor signaling that results in thepathophysiology of the disorder, or in the homeostatic response to thedisorder. Thus, the antibodies may find use for example to prevent,treat, or alleviate, diseases or conditions or associated symptoms orsequelae, of the endocrine system, the central nervous system, theperipheral nervous system, the cardiovascular system, the pulmonarysystem, and the gastrointestinal system, while reducing and oreliminating one or more of the unwanted side effects associated with thecurrent treatments. Glucagon related metabolic disorders include, butare not limited to, type 1 and type 2 diabetes, diabetic ketoacidosis,hyperglycemia, hyperglycemic hyperosmolar syndrome, perioperativehyperglycemia, hyperglycemia in the intensive care unit patient,hyperinsulinemia, postprandial hyperglycemia, hyperglycemia associatedwith burns, or myocardial infarct, or other cardiac problems/conditions,impaired fasting glucose (IFG), metabolic syndrome, hyper-/hypokalemia,poor LDL/HDL ratio, eating disorders, weight gain, obesity as aconsequence of diabetes, pediatric diabetes, gestational diabetes,diabetic late complications, micro-/macroalbuminuria, nephropathy,retinopathy, neuropathy, diabetic foot ulcers, wound healing, impairedglucose tolerance (IGT), insulin resistance syndromes, syndrome X,glucagonomas, gastrointestinal disorders, obesity, diabetes as aconsequence of obesity, etc. The present invention further provides; amethod of treating conditions resulting from excessive glucagon in amammal; a method of inhibiting the glucose elevating effects of glucagonin a mammal; a method of inhibiting a glucagon mediated cellularresponse in a mammal, or a method of reducing the glycemic level in amammal comprising administering to a mammal in need of such treatment aglucagon inhibiting amount of an anti-GCG antibody or a biologicallyactive fragment thereof.

In one embodiment, the antibodies of the present invention may be usedto prevent the onset of hyperglycemia, for example, stress-inducedhyperglycemia, or to reduce the likelihood of onset of hyperglycemia ina patient, or may be used to reduce the severity of a disease orcondition resulting in part from elevated blood glucose levels. In sucha setting, it is envisioned that the anti-GCG antibodies of the presentinvention may be used in an acute setting, either alone, or inconjunction with a second therapeutic agent, for example, insulin. Inone embodiment, the anti-GCG antibodies may be used as chronic therapyto lower blood glucose or to maintain normal levels of blood glucosewhen used alone or with a second therapeutic agent, such as insulin.

The present antibodies are effective in lowering blood glucose, both inthe fasting and the postprandial stage. In certain embodiments of theinvention, the present antibodies are used for the preparation of apharmaceutical composition for the treatment of type 2 diabetes. In yeta further embodiment of the invention the present antibodies are usedfor the preparation of a pharmaceutical composition for the delaying orprevention of the progression from impaired glucose tolerance to type 2diabetes. In yet another embodiment of the invention the presentantibodies are used for the preparation of a pharmaceutical compositionfor the delaying or prevention of the progression from non-insulinrequiring diabetes to insulin requiring diabetes. In a furtherembodiment of the invention the present antibodies are used for thepreparation of a pharmaceutical composition for the treatment of type 1diabetes.

In another embodiment, the antibodies of the invention are useful fortreating stress hyperglycemia resulting from exposure of a patient toany one or more stressors, or to a stress-inducing stimulus, or to aglucose elevating stimulus selected from the group consisting ofpre-existing type 1 or type 2 diabetes, hypertonic dehydration, infusionof catecholamine pressors, parenteral nutrition, enteral nutrition,glucocorticoid therapy, obesity, aging, excessive dextroseadministration, pancreatitis, sepsis, stroke, a myocardial infarct orother cardiac condition, burns, traumatic head injury, hypothermia,hypoxemia, uremia, cirrhosis, anesthesia, pre-operative orpost-operative hospital stays (peri-operative hyperglycemia), admissionto an emergency room, a trauma center, or an intensive care unit,prolonged hospital stays, surgical procedures, an infection and achronic illness.

It is envisioned that the antibodies of the invention may be used in anacute setting (for short term use), or for longer term (chronic) use.Such treatment may be accompanied by insulin therapy, or other glucoselowering therapy.

Combination Therapies

Combination therapies may include an anti-GCG antibody of the inventionand any additional therapeutic agent that may be advantageously combinedwith an antibody of the invention, or with a biologically activefragment of an antibody of the invention.

For example, a second therapeutic agent may be employed to aid infurther lowering of glucose levels, or to reduce at least one symptom ina patient suffering from a disease or condition characterized by highblood glucose levels, such as diabetes mellitus. Such a second agent maybe selected from, for example, a glucagon receptor antagonist (asdescribed in, for example, U.S. Pat. No. 8,545,847), or another GCGantagonist (e.g. another different anti-glucagon antibody or ananti-GCGR antibody or small molecule inhibitor of glucagon or GCGR), ormay include other therapeutic moieties useful for treating diabetes, orother diseases or conditions associated with, or resulting from elevatedblood glucose levels, or impaired glucose metabolism, or agents usefulfor treating any long term complications associated with elevated and/oruncontrolled blood glucose levels. These agents include biguanides,which decrease glucose production in the liver and increase sensitivityto insulin (e.g. metformin), or sulfonylureas, which stimulate insulinproduction (e.g. glyburide, glipizide). Additional treatments directedat maintaining glucose homeostasis including PPAR gamma agonists, suchas the thiazolidinediones, which act as insulin sensitizers (e.g.pioglitazone, rosiglitazone); and alpha glucosidase inhibitors, whichslow starch absorption and glucose production (e.g. acarbose,voglibose). Additional treatments include injectable treatments such asa glucagon-like peptide 1 (GLP-1) analogue or agonist, for example,BYETTA® (exenatide) or VICTOZA® (liraglutide). Another treatment may bewith SYMLIN® (pramlintide), which is an analogue of amylin, a smallpeptide hormone that is released into the bloodstream by the β cells ofthe pancreas along with insulin, after a meal. By augmenting endogenousamylin, pramlintide aids in the absorption of glucose by slowing gastricemptying, promoting satiety via hypothalamic receptors (differentreceptors than for GLP-1), and inhibiting inappropriate secretion ofglucagon. Other compounds that may be used in combination with theantibodies of the invention include dipeptidyl peptidase IV inhibitors,(DPP-4 inhibitors), which reduce glucagon and blood glucose levels. Themechanism of DPP-4 inhibitors is to increase incretin levels (GLP-1 andGIP), which inhibit glucagon release, which in turn increases insulinsecretion, decreases gastric emptying, and decreases blood glucoselevels. Examples of DPP-4 inhibitors include saxagliptin (ONGLYZA®),sitaliptin (JANUVIA®), and vildagliptin (GALVUS®). Other compounds thatmay be used in combination with the antibodies of the invention includesodium-glucose co-transporter 2 (SGLT2) inhibitors, which block thereabsorption of glucose in the kidney, increase glucose excretion andlower blood glucose levels. Examples of SGLT2 inhibitors includeINVOKANA™ (canagliflozin), FORXIGA® (dapagliflozin), empagliflozin,ipragliflozin and tofogliflozin.

In certain other embodiments, the composition may include a second agentselected from the group consisting of non-sulfonylurea secretagogues,insulin, insulin analogs, exendin-4 polypeptides, beta 3 adrenoceptoragonists, statins and statin-containing combinations, cholesterolabsorption inhibitors, LDL-cholesterol antagonists, cholesteryl estertransfer protein antagonists, endothelin receptor antagonists, growthhormone antagonists, insulin sensitizers, amylin mimetics or agonists,cannabinoid receptor antagonists, glucagon-like peptide-1 agonists,melanocortins, melanin-concentrating hormone receptor agonists, SNRIs,and protein tyrosine phosphatase inhibitors.

In certain other embodiments, combination therapy may includeadministration of a second agent to counteract any potential sideeffect(s) resulting from administration of an antibody of the invention,if such side effect(s) occur. For example, in the event that any of theanti-GCG antibodies increases lipid or cholesterol levels, it may bebeneficial to administer a second agent to lower lipid or cholesterollevels, using an agent such as a HMG-CoA reductase inhibitor (forexample, a statin such as atorvastatin, (LIPITOR®), fluvastatin(LESCOL®), lovastatin (MEVACOR®), pitavastatin (LIVALO®), pravastatin(PRAVACHOL®), rosuvastatin (CRESTOR®) and simvastatin (ZOCOR®).Alternatively, the antibodies of the invention may be combined with anagent such as VYTORIN®, which is a preparation of a statin and anotheragent—such as ezetimibe/simvastatin.

In certain embodiments, it may be beneficial to administer theantibodies of the invention in combination with any one or more of thefollowing: (1) niacin, which increases lipoprotein catabolism; (2)fibrates or amphipathic carboxylic acids, which reduce low-densitylipoprotein (LDL) level, improve high-density lipoprotein (HDL) and TGlevels, and reduce the number of non-fatal heart attacks; and (3)activators of the LXR transcription factor that plays a role incholesterol elimination such as 22-hydroxycholesterol, or a statin witha bile resin (e.g., cholestyramine, colestipol, colesevelam), a fixedcombination of niacin plus a statin (e.g., niacin with lovastatin); orwith other lipid lowering agents such as omega-3-fatty acid ethyl esters(for example, omacor).

Furthermore, the second therapeutic agent can be one or more otherinhibitor/antagonist of glucagon or an inhibitor/antagonist of theglucagon receptor (GCGR), as well as inhibitors of other molecules, suchas angiopoietin-like protein 3 (ANGPTL3), angiopoietin-like protein 4(ANGPTL4), angiopoietin-like protein 5 (ANGPTL5), angiopoietin-likeprotein 6 (ANGPTL6), angiopoietin-like protein 8 (ANGPTL8), which areinvolved in lipid metabolism, in particular, cholesterol and/ortriglyceride homeostasis. Inhibitors of these molecules include smallmolecules and/or antibodies that specifically bind to these moleculesand block their activity.

In certain embodiments, it may be beneficial to administer theantibodies of the invention in combination with an antibody that acts tolower lipid or cholesterol levels, such as, but not limited to, forexample, any anti-PCSK9 (proprotein convertase subtilisin/kexin type 9)antibody, such as those described in US2010/0166768 (now U.S. Pat. No.8,062,640). Other anti-PCSK9 antibodies are described in US2010/0040611,US2010/0041102, US2010/0040610, US2010/0113575, US2009/0232795,US2009/0246192, US2010/0233177, US2009/0142352, US2009/0326202,US2010/0068199, US2011/0033465, US2011/0027287, US2010/0150937,US2010/0136028 and WO2009/055783.

In certain embodiments, it may be beneficial to administer the anti-GCGantibodies of the invention in combination with a nucleic acid thatinhibits the activity of PCSK9 (proprotein convertase subtilisin/kexintype 9), such as an antisense molecule, a double stranded RNA, or asiRNA molecule. Exemplary nucleic acid molecules that inhibit theactivity of PCSK9 are described in US2011/0065644, US2011/0039914,US2008/0015162 and US2007/0173473.

The additional therapeutically active component(s) may be administeredprior to, concurrent with, or after the administration of the anti-GCGantibody of the present invention. For purposes of the presentdisclosure, such administration regimens are considered theadministration of an anti-GCG antibody “in combination with” a secondtherapeutically active component.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of an anti-GCG antibody (or a pharmaceutical compositioncomprising a combination of an anti-GCG antibody and any of theadditional therapeutically active agents mentioned herein) may beadministered to a subject over a defined time course. The methodsaccording to this aspect of the invention comprise sequentiallyadministering to a subject multiple doses of an anti-GCG antibody of theinvention. As used herein, “sequentially administering” means that eachdose of anti-GCG antibody is administered to the subject at a differentpoint in time, e.g., on different days separated by a predeterminedinterval (e.g., hours, days, weeks or months). The present inventionincludes methods which comprise sequentially administering to thepatient a single initial dose of an anti-GCG antibody, followed by oneor more secondary doses of the anti-GCG antibody, and optionallyfollowed by one or more tertiary doses of the anti-GCG antibody.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the anti-GCG antibody ofthe invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount ofanti-GCG antibody, but generally may differ from one another in terms offrequency of administration. In certain embodiments, however, the amountof anti-GCG antibody contained in the initial, secondary and/or tertiarydoses varies from one another (e.g., adjusted up or down as appropriate)during the course of treatment. In certain embodiments, two or more(e.g., 2, 3, 4, or 5) doses are administered at the beginning of thetreatment regimen as “loading doses” followed by subsequent doses thatare administered on a less frequent basis (e.g., “maintenance doses”).

In certain exemplary embodiments of the present invention, eachsecondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2,2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½,12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½,20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more)weeks after the immediately preceding dose. The phrase “the immediatelypreceding dose,” as used herein, means, in a sequence of multipleadministrations, the dose of anti-GCG antibody, which is administered toa patient prior to the administration of the very next dose in thesequence with no intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an anti-GCG antibody. For example, in certain embodiments, only asingle secondary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondarydoses are administered to the patient. Likewise, in certain embodiments,only a single tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient. The administration regimen may becarried out indefinitely over the lifetime of a particular subject, oruntil such treatment is no longer therapeutically needed oradvantageous.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks or 1 to 2 months after the immediately preceding dose.Similarly, in embodiments involving multiple tertiary doses, eachtertiary dose may be administered at the same frequency as the othertertiary doses. For example, each tertiary dose may be administered tothe patient 2 to 12 weeks after the immediately preceding dose. Incertain embodiments of the invention, the frequency at which thesecondary and/or tertiary doses are administered to a patient can varyover the course of the treatment regimen. The frequency ofadministration may also be adjusted during the course of treatment by aphysician depending on the needs of the individual patient followingclinical examination.

Diagnostic Uses of the Antibodies

The anti-GCG antibodies of the present invention may also be used todetect and/or measure GCG in a sample, e.g., for diagnostic purposes.For example, an anti-GCG antibody, or fragment thereof, may be used todiagnose a condition or disease characterized by aberrant expression(e.g., over-expression, under-expression, lack of expression, etc.) ofGCG. Exemplary diagnostic assays for GCG may comprise, e.g., contactinga sample, obtained from a patient, with an anti-GCG antibody of theinvention, wherein the anti-GCG antibody is labeled with a detectablelabel or reporter molecule or used as a capture ligand to selectivelyisolate GCG protein from patient samples. Alternatively, an unlabeledanti-GCG antibody can be used in diagnostic applications in combinationwith a secondary antibody which is itself detectably labeled. Thedetectable label or reporter molecule can be a radioisotope, such as ³H,¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent or chemiluminescent moiety such asfluorescein isothiocyanate, or rhodamine; or an enzyme such as alkalinephosphatase, β-galactosidase, horseradish peroxidase, or luciferase.Specific exemplary assays that can be used to detect or measure GCG in asample include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in GCG diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient, which contains detectable quantities of GCG protein, orfragments thereof, under normal or pathological conditions. Generally,levels of GCG in a particular sample obtained from a healthy patient(e.g., a patient not afflicted with a disease or condition associatedwith abnormal GCG levels or activity) will be measured to initiallyestablish a baseline, or standard, level of GCG. This baseline level ofGCG can then be compared against the levels of GCG measured in samplesobtained from individuals suspected of having a GCG related disease orcondition, or symptoms associated with such disease or condition.

EXAMPLES

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims. Efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isaverage molecular weight, temperature is in degrees Centigrade, andpressure is at or near atmospheric.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, preferred methods and materials are now described. Allpublications mentioned herein are incorporated herein by reference intheir entirety.

Example 1 Generation of Anti-GCG Antibodies

Anti-GCG antibodies were obtained by immunizing a VELOCIMMUNE® mouse(i.e., an engineered mouse comprising DNA encoding human immunoglobulinheavy and kappa light chain variable regions) with an immunogencomprising human GCG. The antibody immune response was monitored by aGCG-specific immunoassay. When a desired immune response was achieved,several fully human anti-GCG antibodies were generated fromantigen-positive B cells as described in US 200710280945A1, incorporatedby reference herein in its entirety.

Certain biological properties of the exemplary anti-GCG antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

Example 2 Heavy and Light Chain Variable Region Amino Acid and NucleicAcid Sequences

Table 1 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions and CDRs of selected anti-GCG antibodies ofthe invention. The corresponding nucleic acid sequence identifiers areset forth in Table 2.

TABLE 1 Amino Acid Sequence Identifiers Antibody SEQ ID NOs: DesignationHCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H059P 2 4 6 8 10 12 1416 H4H10223P 18 20 22 24 26 28 30 32 H4H10231P 34 36 38 40 42 44 46 48H4H10232P 50 52 54 56 58 60 62 64 H4H10236P 66 68 70 72 74 76 78 80H4H10237P 82 84 86 88 90 92 94 96 H4H10238P 98 100 102 104 106 108 110112 H4H10250P 114 116 118 120 122 124 126 128 H4H10256P 130 132 134 136138 140 142 144 H4H10270P 146 148 150 152 154 156 158 160

TABLE 2 Nucleic Acid Sequence Identifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H059P 1 3 57 9 11 13 15 H4H10223P 17 19 21 23 25 27 29 31 H4H10231P 33 35 37 39 4143 45 47 H4H10232P 49 51 53 55 57 59 61 63 H4H10236P 65 67 69 71 73 7577 79 H4H10237P 81 83 85 87 89 91 93 95 H4H10238P 97 99 101 103 105 107109 111 H4H10250P 113 115 117 119 121 123 125 127 H4H10256P 129 131 133135 137 139 141 143 H4H10270P 145 147 149 151 153 155 157 159

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H1H,” “H1M,” “H2M,” etc.), followed by anumerical identifier (e.g. “10223,” “10231,” “10232,” etc.), followed bya “P” or “N” suffix, as shown in Tables 1 and 2. Thus, according to thisnomenclature, an antibody may be referred to herein as, e.g.,“H4H10270P”, etc. The H4H prefix on the antibody designations usedherein indicate the particular Fc region isotype of the antibody. Forexample, an “H4H” antibody has a human IgG4 Fc, an “H1M” antibody has amouse IgG1 Fc, and an “H2M” antibody has a mouse IgG2 Fc, (all variableregions are fully human as denoted by the first ‘H’ in the antibodydesignation). As will be appreciated by a person of ordinary skill inthe art, an antibody having a particular Fc isotype can be converted toan antibody with a different Fc isotype (e.g., an antibody with a mouseIgG1 Fc can be converted to an antibody with a human IgG4, etc.), but inany event, the variable domains (including the CDRs)—which are indicatedby the numerical identifiers shown in Tables 1 and 2—will remain thesame, and the binding properties are expected to be identical orsubstantially similar regardless of the nature of the Fc domain.

Example 2 Variable Gene Utilization Analysis

To analyze the structure of antibodies produced, the nucleic acidsencoding antibody variable regions were cloned and sequenced. From thenucleic acid sequence and predicted amino acid sequence of theantibodies, gene usage was identified for each Heavy Chain VariableRegion (HCVR) and Light Chain Variable Region (LCVR). Table 3 sets forththe gene usage for selected antibodies in accordance with the invention.

TABLE 3 HCVR LCVR AbPID VH D JH VK JK H1H059P 3-30 2-2 4 4-1 1 H4H10223P3-30 3-3 4 4-1 2 H4H10231P 3-30 3-3 4 4-1 2 H4H10232P 3-30 3-3 4 4-1 2H4H10236P 3-30 3-3 4 4-1 2 H4H10237P 3-30 3-3 4 4-1 2 H4H10238P 3-30 3-34 4-1 2 H4H10250P 3-30 3-3 4 4-1 2 H4H10256P 3-30 3-3 4 4-1 2 H4H10270P3-30 3-3 4 4-1 2

Example 3 Surface Plasmon Resonance Derived Binding Affinities andKinetic Constants of Human Monoclonal Anti-GCG Antibodies

Equilibrium dissociation constants (K_(D)) values for purified anti-GCGantibodies binding to human GCG were determined using a real-timesurface plasmon resonance biosensor assay on a Biacore 4000 instrument.The Biacore sensor surface was derivatized with a monoclonal mouseanti-human Fc antibody (GE Healthcare, # BR-1008-39) to capture eachanti-GCG monoclonal antibody. Different concentrations of human GCG(Phoenix Pharmaceuticals, #028-02) were injected over the anti-GCGmonoclonal antibody captured surface at a flow rate of 30 μL/min.Binding of GCG to the captured monoclonal antibodies was monitored for 4minutes while the dissociation was monitored for 10 minutes in HBSTrunning buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v/vSurfactant P20). Experiments were performed at 25° C. and 37° C.

Kinetic association (k_(a)) and dissociation (k_(d)) rate constants weredetermined by processing and fitting the data to a 1:1 binding modelusing Scrubber 2.0c curve fitting software. Binding dissociationequilibrium constants (K_(D)) and dissociative half-lives (t½) werecalculated from the kinetic rate constants as:

${{K_{D}(M)} = {{kd}/{ka}}},{{{and}\mspace{14mu} t\;{1/2}\mspace{14mu}\left( \min \right)} = \frac{\ln(2)}{60*{kd}}}$

Binding kinetics parameters for different anti-GCG monoclonal antibodiesbinding to human GCG reagents at 25° C. and 37° C. are shown in Tables 4and 5, respectively.

TABLE 4 Binding kinetics of anti-GCG antibodies binding to human GCG at25° C. Human GCG Binding at 25° C. k_(a) k_(d) K_(D) t½ Antibody (1/Ms)(1/s) (M) (min) H4H10236P 3.08E+06 7.46E−04 2.42E−10 15 H4H10237P4.96E+06 1.03E−03 2.08E−10 11 H4H10238P 3.89E+06 6.20E−04 1.60E−10 19H4H10250P 3.32E+06 9.39E−04 2.83E−10 12 H4H10256P 7.72E+06 5.82E−047.53E−11 20 H4H10270P 4.80E+06 7.32E−04 1.53E−10 16 H4H10223P 9.36E+065.41E−04 5.78E−11 21 H4H10231P 7.39E+06 6.85E−04 9.27E−11 17 H4H10232P3.19E+06 2.31E−04 7.24E−11 50 H1H059P 2.50E+06 1.78E−03 7.15E−10 6

TABLE 5 Binding kinetics of anti-GCG antibodies binding to human GCG at37° C. Human GCG Binding at 37° C. k_(a) k_(d) K_(D) t½ Antibody (1/Ms)(1/s) (M) (min) H4H10236P 4.25E+06 8.80E−03 2.08E−09 1 H4H10237P7.53E+06 6.83E−03 9.07E−10 2 H4H10238P 4.94E+06 2.74E−03 5.55E−10 4H4H10250P 5.58E+06 7.08E−03 1.27E−09 2 H4H10256P 7.90E+06 2.48E−033.14E−10 5 H4H10270P 6.77E+06 3.78E−03 5.59E−10 3 H4H10223P 6.40E+062.23E−03 3.47E−10 5 H4H10231P 2.40E+06 2.45E−03 1.01E−09 5 H4H10232P4.61E+06 1.47E−03 3.18E−10 8 H1H059P 4.90E+06 2.36E−02 4.78E−09 0.5

As shown in Table 4, all of the anti-GCG antibodies bound human GCG at25° C. with K_(D) values ranging from 57.8 pM to about 715 pM. As shownin Table 5, all of the anti-GCG antibodies bound human GCG at 37° C.with K_(D) values ranging from 314 pM to about 4.78 nM.

Example 4 GCG Bioassay with HEK293/CRE-Luciferase/hGCGR Cells

GCGR is a G-protein coupled receptor and its ligand, glucagon (GCG),stimulates adenylyl cyclase activity through G

s and phosphoinositol turnover through Gq (Jiang and Zhang, (2003),Amer. J. Physiol.—Endocrinol. And Metabolism, 1 April, Vol. 284, No.E671-E678). A bioassay was developed to detect activation through G

s, subsequent elevation of cAMP levels, and transcriptional activation.HEK293 cell lines were generated to stably express full-length humanGCGR (hGCGR; amino acids 1-477 of accession number NP000151.1) alongwith a luciferase reporter [cAMP response element (CRE,4×)-luciferase-IRES-GFP]. The stable cell line (HEK293/CRE-luc/hGCGRcells) was isolated and maintained in DMEM containing 10% FBS, NEAA, andpenicillin/streptomycin/L-glutamine.

For the bioassay, HEK293/CRE-luc/hGCGR cells were seeded onto 96-wellassay plates at 10,000 cells/well in low serum media (OPTIMEM containing0.1% FBS), and incubated at 37° C. in 5% CO₂ overnight. The next day,human GCG (Phoenix Pharmaceuticals, #028-02) was serially diluted at 1:3from 10 nM to 0.0002 nM and added to cells. A control containingdilution buffer but no GCG was also added to one sample of cells. Tomeasure inhibition, anti-GCG antibodies were serially diluted at 1:3from 100 nM to 0.002 nM and added to cells including a control samplecontaining no antibody with a constant concentration of 40 pM GCG. After5.5 hours of incubation at 37° C. in 5% CO₂, luciferase activity wasdetected using a Victor X plate reader (Perkin Elmer). The results wereanalyzed using nonlinear regression (4-parameter logistics) with Prism 6software (GraphPad).

As shown in Table 6, all 10 anti-GCG antibodies tested inhibited allactivation of HEK293/CRE-luc/hGCGR cells by 40 pM of GCG with IC₅₀values ranging from 7.3 pM to 910 pM. The isotype control antibody didnot demonstrate any measurable blockade and GCG activated theHEK293/CRE-luc/hGCGR cell line with an EC₅₀ value of 53 pM.

TABLE 6 Inhibition of GCG activation of HEK293/CRE-luc/hGCGR cells byanti-GCG antibodies GCG EC₅₀ [M] 5.3E−11 GCG constant used to testantibodies 40 pM Antibodies IC₅₀ [M] H4H10223P 9.5E−12 H4H10231P 7.3E−12H4H10232P 2.3E−11 H4H10236P 2.5E−10 H4H10237P 3.4E−11 H4H10238P 1.8E−11H4H10250P 3.4E−11 H4H10256P 1.8E−11 H4H10270P 2.2E−11 H1H059P 9.1E−10Isotype control Non-blocker antibody

Example 5 The Effect of a Single Dose of H4H10223P in Diet-InducedObesity Mouse in an In Vivo Model of Type 2 Diabetes

The effects of a single dose of a specific anti-GCG antibody of theinvention, H4H10223P, on blood glucose levels was determined in adiet-induced obesity (D10) mouse model of type 2 diabetes. The DIO modelis developed by feeding mice a high fat (60% kcal) diet (HFD) startingat 5˜6 weeks of age. After approximately 6 weeks on the HFD, micedevelop metabolic abnormalities, including insulin resistance, glucoseintolerance, and obesity. Twenty-three 3-month-old male C57BL/6 mice(Taconic farms, Inc., B6-M) were placed on a HFD at 5 weeks of age andwere kept on the diet for the subsequent 4 months. At 7 months of age,the mice were divided into 4 groups of 2 to 4 animals. Each groupreceived a single subcutaneous injection of H4H10223P at 3 mg/kg (n=5),10 mg/kg (n=3), or 30 mg/kg (n=2) or an isotype control antibody, whichdoes not bind to any known mouse protein, at 30 mg/kg (n=5). At days 2,4, 7, and 9 following antibody dosing, tail bleeds were collected frommice. Blood glucose levels from the tail bleed samples were determinedusing ACCU-CHEK® Compact Plus (Roche, 075177294001). The percentreduction in blood glucose from the mean blood glucose levels of thecontrol group was calculated for each animal at each time point. Theaverage percent reduction in blood glucose was calculated for eachtreatment group. Table 7 summarizes the mean blood glucose levels ofeach treatment group. Results, expressed as (mean±SEM) of percent bloodglucose reduction, are shown in Table 8.

As shown in Tables 7 and 8, mice treated with a single dose of H4H10223Pat 30 mg/kg exhibited significant reductions in blood glucose levels atall time points measured compared to mice injected with isotype controlantibody. Mice treated with a single dose of H4H10223P at 3 mg/kgexhibited a significant reduction in blood glucose levels at day 2compared to mice injected with isotype control antibody, but there wereno significant reductions at this dosage observed at the other timepoints measured. Mice treated with a single dose of H4H10223P at 10mg/kg exhibited a significant reduction in blood glucose levels at day 2compared to mice injected with isotype control antibody, and there werereduction trends at this dosage observed at the other time pointsmeasured.

TABLE 7 Blood glucose levels (mg/dL) from each treatment group Bloodglucose level (mg/dL) Time Control H4H10223P (days) 30 mg/kg 3 mg/kg 10mg/kg 30 mg/kg 0 210 ± 6  209 ± 8 209 ± 10  209 ± 12 2 192 ± 8  172 ± 7164 ± 7  142 ± 5 4 168 ± 10 181 ± 6 152 ± 18 137 ± 2 7 178 ± 12 173 ± 8169 ± 3  149 ± 1 9 174 ± 10 179 ± 4 170 ± 30 155 ± 7

TABLE 8 Percent reduction in blood glucose levels from each treatmentgroup as compared to isotype control treatment Blood glucose reduction(%) Time H4H10223P (days) 3 mg/kg 10 mg/kg 30 mg/kg 2 10 ± 3 14 ± 4  26± 2 4 −8 ± 3 9 ± 11 18 ± 1 7  3 ± 5 5 ± 2  16 ± 1 9 −3 ± 3 2 ± 17 11 ± 4

Example 6 The Effect of Multiple Doses (Chronic Administration) ofAnti-GCG Antibodies H4H10223P and H4H10231P in an In Vivo Diet-InducedObesity Mouse Model of Type 2 Diabetes

The chronic effects of specific anti-GCG antibodies of the invention,H4H10223P and H4H10231P, on blood glucose levels were determined in adiet-induced obesity (DIO) mouse model of type 2 diabetes. As notedabove, the DIO model is developed by feeding mice a high fat (60% kcal)diet (HFD) starting at approximately 5 to 6 weeks of age.

In one experiment (Study 1), Thirteen male C57BL/6 mice (Taconic Farms,Inc., # B6-M) were placed on a HFD at 5 weeks of age and were kept onthe diet for the subsequent 21 weeks. At 26 weeks of age, the mice weredivided into 2 groups of 5 or 8 animals. Each group receivedsubcutaneous injections of H4H10223P (n=5; 30 mg/kg), or an isotypecontrol antibody (n=8; 30 mg/kg), which does not bind to any known mouseprotein, every five days. Two, 6, 9, 13, 16 and 20 days following theinitial antibody dosing, tail bleeds were collected from mice. Bloodglucose levels from the tail bleed samples were determined usingACCU-CHEK® Compact Plus (Roche, #075177294001). The percent reduction inblood glucose from the mean blood glucose levels of the control groupwas calculated for each animal at each time point. The mean percentreduction in blood glucose was calculated for the H4H10223P group ateach time point.

Table 9 summarizes the mean blood glucose levels of each treatment groupand mean percent blood glucose reductions of the H4H10223P group.

In another experiment (Study 2), thirty-one male C57BL/6 mice wereplaced on a HFD at 5 weeks of age and were kept on the diet for thesubsequent 11 weeks. At 16 weeks of age, the mice were divided into 4groups of 7 or 8 animals. Each group received subcutaneous injections ofH4H10223P (30 mg/kg; n=8), H4H10231P (5 or 30 mg/kg; n=8), or theisotype control antibody (30 mg/kg; n=7), every five days. One, 4, 7,11, 15 and 20 days following the initial antibody dosing, tail bleedswere collected from mice.

Table 10 summarizes the mean blood glucose levels of each treatmentgroup.

Table 11 summarizes the mean percent reductions in blood glucose fromthe mean blood glucose levels of the control group for each treatmentgroup.

TABLE 9 (Study 1) Blood glucose levels of each treatment group andpercent reductions in blood glucose levels as compared to the controlgroup for the H4H10223P group H4H10223P Control (30 mg/kg) Blood Bloodglucose glucose Time level level % (days) (mg/dL) (mg/dL) Reduction 0211 ± 7 211 ± 6  0 ± 3 2  212 ± 24 146 ± 9 31 ± 4 6 200 ± 7 154 ± 6 23 ±3 9 174 ± 8 148 ± 7 15 ± 4 13  182 ± 12 144 ± 5 21 ± 3 16 158 ± 5 122 ±5 23 ± 3 20  155 ± 11 139 ± 9 10 ± 6

TABLE 10 (Study 2) Blood glucose levels (mg/dL) of each treatment groupTime H4H10231P H4H10231P (days) Control H4H10223P (5 mg/kg) (30 mg/kg) 0 225 ± 10  225 ± 10  225 ± 10  225 ± 10 1  206 ± 11 214 ± 5 172 ± 3 192± 7 4 194 ± 4 152 ± 7 156 ± 5 141 ± 5 7 215 ± 8 191 ± 6 181 ± 5 167 ± 611 199 ± 6 164 ± 8 160 ± 8 149 ± 6 15  227 ± 10  222 ± 14  219 ± 17 191± 5 20 204 ± 5 183 ± 9 180 ± 5 171 ± 7

TABLE 11 (Study 2) Percent reductions in blood glucose levels ascompared to the control group for each treatment group Time H4H10231PH4H10231P (days) H4H10223P (5 mg/kg) (30 mg/kg) 0  0 ± 5  0 ± 4  0 ± 5 1−4 ± 2 17 ± 2  7 ± 3 4 22 ± 3 20 ± 3 27 ± 2 7 11 ± 3 16 ± 2 23 ± 3 11 18± 4 20 ± 4 25 ± 3 15  3 ± 6  4 ± 8 16 ± 2

RESULTS

As shown in Tables 9 and 10, H4H10223P and H4H10231P decreased bloodglucose levels significantly. Maximum percent reductions in bloodglucose were 31% for the H4H10223P group in study 1, 22% for theH4H10223P group in study 2, and 27% for the H4H10231P group in study 2(Tables 9 and 11).

What is claimed is:
 1. An isolated antibody or antigen-binding fragmentthereof that specifically binds to glucagon (GCG) and neutralizes GCGactivity, wherein the antibody or antigen-binding fragment thereofcomprises: (a) three heavy chain complementarity determining regions(HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region(HCVR) amino acid sequence selected from the group consisting of SEQ IDNOs: 2, 18, 34, 50, 66, 82, 98, 114, 130 and 146; and (b) three lightchain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chainvariable region (LCVR) amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138 and 154.2. The isolated antibody or antigen-binding fragment thereof of claim 1,wherein the antibody or antigen-binding fragment comprises an HCVRhaving an amino acid sequence selected from the group consisting of SEQID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130 and 146; and a LCVR havingan amino acid sequence selected from the group consisting of SEQ ID NOs:10, 26, 42, 58, 74, 90, 106, 122, 138 and
 154. 3. The isolated antibodyor antigen-binding fragment thereof of claim 2, comprising a HCVR/LCVRamino acid sequence pair selected from the group consisting of SEQ IDNOs: 2/10; 18/26; 34/42; 50/58; 66/74; 82/90; 98/106; 114/122; 130/138and 146/154.
 4. The isolated antibody or antigen-binding fragmentthereof of claim 1, comprising: (a) a HCDR1 domain having an amino acidsequence selected from the group consisting of SEQ ID NOs: 4, 20, 36,52, 68, 84, 100, 116, 132 and 148; (b) a HCDR2 domain having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 6, 22,38, 54, 70, 86, 102, 118, 134 and 150; (c) a HCDR3 domain having anamino acid sequence selected from the group consisting of SEQ ID NOs: 8,24, 40, 56, 72, 88, 104, 120, 136 and 152; (d) a LCDR1 domain having anamino acid sequence selected from the group consisting of SEQ ID NOs:12, 28, 44, 60, 76, 92, 108, 124, 140 and 156; (e) a LCDR2 domain havingan amino acid sequence selected from the group consisting of SEQ ID NOs:14, 30, 46, 62, 78, 94, 110, 126, 142, and 158; and (f) a LCDR3 domainhaving an amino acid sequence selected from the group consisting of SEQID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144 and
 160. 5. The isolatedantibody or antigen-binding fragment thereof of claim 1, comprising: (a)a HCDR1 domain comprising the amino acid sequence of SEQ ID NO: 20; (b)a HCDR2 domain comprising the amino acid sequence of SEQ ID NO: 22; (c)a HCDR3 domain comprising the amino acid sequence of SEQ ID NO: 24; (d)a LCDR1 domain comprising the amino acid sequence of SEQ ID NO: 28; (e)a LCDR2 domain comprising the amino acid sequence of SEQ ID NO: 30; and(f) a LCDR3 domain comprising the amino acid sequence of SEQ ID NO: 32.6. The isolated antibody or antigen-binding fragment thereof of claim 1,comprising: (a) a HCDR1 domain comprising the amino acid sequence of SEQID NO: 36; (b) a HCDR2 domain comprising the amino acid sequence of SEQID NO: 38; (c) a HCDR3 domain comprising the amino acid sequence of SEQID NO: 40; (d) a LCDR1 domain comprising the amino acid sequence of SEQID NO: 44; (e) a LCDR2 domain comprising the amino acid sequence of SEQID NO: 46; and (f) a LCDR3 domain comprising the amino acid sequence ofSEQ ID NO:
 48. 7. An isolated monoclonal antibody or antigen-bindingfragment thereof of claim 1, wherein the antibody or fragment thereofexhibits one or more of the following characteristics: (a) is a fullyhuman monoclonal antibody; (b) binds human GCG at 25° C. with a K_(D)ranging from about 50 pM to about 750 pM and less than from about 300 pMto about 5 nM at 37° C. as measured by surface plasmon resonance; (c)lowers blood glucose levels by at least about 10% when administered to amammal as a single dose, or as multiple doses of less than about 40mg/kg; or (d) inhibits GCG-mediated activation of cells expressing humanglucagon receptor (GCGR) with an IC₅₀ ranging from about 7 pM to about950 pM.
 8. The isolated antibody or antigen-binding fragment thereof ofclaim 7, wherein the antibody lowers blood glucose levels by about 10%to about 31% when administered to a mammal as a single dose, or asmultiple doses ranging from about 3 mg/kg to about 30 mg/kg.
 9. Theisolated antibody or antigen-binding fragment thereof of claim 7,wherein the antibody or an antigen-binding fragment thereof reducesblood glucose levels in a mammal when administered subcutaneously,intravenously, or intramuscularly.
 10. The isolated antibody orantigen-binding fragment thereof of claim 7, wherein the antibody or anantigen-binding fragment thereof reduces blood glucose levels in amammal suffering from a condition or disease associated with, orcharacterized in part by high blood glucose levels.
 11. The isolatedantibody or antigen-binding fragment thereof of claim 10, wherein thecondition or disease associated with, or characterized in part by highblood glucose levels is selected from the group consisting of diabetes,impaired glucose tolerance, obesity, hyperglycemia, hyperglycemichyperosmolar syndrome, perioperative hyperglycemia, hyperglycemia in theintensive care unit patient, hyperinsulinemia, the metabolic syndrome,insulin resistance syndrome and impaired fasting glucose.
 12. Apharmaceutical composition comprising a therapeutically effective amountof an isolated antibody or antigen-binding fragment thereof that bindsto GCG according to claim 1 and a pharmaceutically acceptable carrier ordiluent.
 13. An isolated antibody or antigen-binding fragment thereofthat competes for specific binding to GCG with an antibody orantigen-binding fragment comprising heavy and light chain sequence pairsselected from the group consisting of SEQ ID NOs: 2/10; 18/26; 34/42;50/58; 66/74; 82/90; 98/106; 114/122; 130/138 and 146/154.
 14. Anisolated antibody or antigen-binding fragment thereof that binds to thesame epitope on GCG as a reference antibody comprising an HCVR/LCVRamino acid sequence pair selected from the group consisting of SEQ IDNOs: 2/10; 18/26; 34/42; 50/58; 66/74; 82/90; 98/106; 114/122; 130/138and 146/154.